Wavelength-specific cytotoxic agents

ABSTRACT

A group of hydro-monobenzoporphyrins &#34;green porphyrins&#34; (Gp) having absorption maxima in the range of 670-780 nanometers is useful in treating disorders or conditions which are subject to hematoporphyrin derivative (HPD) treatment in the presence of light, or in treating virus, cells and tissues generally to destroy unwanted targets. The use of the Gp of the invention permits the irradiation to use wavelengths other than those absorbed by blood. The Gp of the invention may also be conjugated to ligands specific for receptor or to specific immunoglobulins or fragments thereof to target specific tissues or cells for the radiation treatment. Use of these materials permits lower levels of drug to be used, thus preventing side reactions which might destroy normal tissues.

CROSS-REFERENCE TO RELATED PATENT APPLICATION

This is a continuation-in-part U.S. Ser. No. 221,161, filed July 19,1988,now U.S. Pat. No. 4,920,143, which is a continuation-in-part ofU.S. Ser. No. 041,680, filed Apr. 23, 1987 no U.S. Pat. No. 4,883,790,which is a continuation-in-part of U.S. Ser. No. 005,204, filed Jan 20,1987, now abandoned.

FIELD OF THE INVENTION

The invention relates to the use of light absorbing compounds to mediatethe destruction of unwanted cells or tissues or, other undesirablematerials by irradiation. Specifically, the invention relates to the useof hydro-monobenzoporphyrin derivatives having absorption maxima in therange 670-780 nanometers to mediate the irradiation of materials to bedestroyed, and to the use of these compounds conjugated totarget-specific ligands, such receptor-specific ligands, orimmunoglobulins or their immunospecific fragments, to focus the effectsof the irradiation on particular targets.

BACKGROUND OF THE INVENTION

The use of hematoporphyrin and its acetylated derivative mixturehematoporphyrin derivative (HPD) systemically, combined withirradiation, for the detection and treatment of malignant cells has, bythis time, some considerable history. HPD is a mixture of porphyrinsincluding hematoporphyrin itself, hyiroxyethyl vinyl deuteroporphyrin,protoporphyrin, and dihematoporphyrin ethers. (See, e.g., "PorphyrinPhotosensitization", Kessel, D., et al, eds. (1983) Plenum Press.)

HPD seems "naturally" capable of localizing in malignant cells. Whenirradiated, it has two properties which make it useful. First, whenirradiated with ultraviolet or visible light, it is capable offluorescence, and thus is useful in diagnostic methods related todetection of malignancy (see, for example, Kessel, et al (supra);Gregory, H.B. Jr., et al, Ann Surq(1968) 167:827-829). More pertinent tothe present invention is the capacity of HPD, when irradiated withvisible light, to exhibit a cytotoxic effect on the cells in which it islocalized (see, for example, Diamond, I., et al, Lancet(1972)2:1175-1177; Dougherty, T.J., et al, Cancer Research(1978) 38:2628-2635;Dougherty, T.J., et al, "The Science of Photo Medicine": (1982) J.D.Regan & J.A. Parrish, eds., pp. 625-638; Dougherty, T.J., et al,"Cancer: Priniciples and Practice of Oncology" (1982) V.T. DeVita Jr.,et al, eds., pp. 1836-1844). Although it has not been definitivelyestablished, the effect of HPD in killing cells seems to be due to theformation of singlet oxygen upon irradiation (Weishaupt, K.R., et al,Cancer Research(1976) 36:2326-2329). Several mechanisms for this effecthave been proposed, and it has recently been shown that the activeingredient in HPD which mediates the cytotoxic effect of visible lightirradiation is the mixture of dihematoporphyrin ethers (DHE) (Dougherty,T.J., et al, "Porphyrin Localization and Treatment of Tumors" (1984) pp.301-314; Dougherty, T.J. CRC Critical Reviews inOncolocy/Hematolocy(1984) 2:83-116).

A purified form of the active component(s) of HPD is obtained byadjustment of pH to cause aggregation and recovery of the aggregate, asdisclosed in U.S. Pat. 4,649,151. The purified form called DHE in thepatent, is marketed under the trademark Photofrin® II and has been usedin a manner completely analogous to HPD.

In addition to in vivo therapeutic and diagnostic protocols for tumorsas described in the above-cited patent, the porphyrins, including HPDand its more purified derivatives, can be used in other in vivo and invitro applications. For example, photosensitizers are useful in thedetection and treatment of atherosclerotic plaques as described in U.S.Pat. Nos. 4,512,762 and 4,577,636. U.S. Pat. Nos. 4,500,507 and4,485,806 describe the use of radiolabeled porphyrin compounds,including HPD, for tumor imaging. U.S. Pat. No. 4,753,958 to theUniversity of California describes the use of topical application ofporphyrin sensitizers for diagnosis and treatment of skin diseases. U.S.Pat. No. 4,748,120 describes the use of photosensitizers in thetreatment of whole blood or blood components. Photochemicaldecontamination treatment of blood and components is also described inU.S. Pat. No. 4,727,027 where the photosensitizer is furocumarin and itsderivatives. In addition, viruses are inactivated in therapeutic proteincompositions in vitro as disclosed in U.S. Pat. No. 4,268,947.

While the treatment of tumors and other undesirable targets with HPDrelies on the intrinsic ability of HPD to localize in malignant cells, aconsiderable improvement and refinement in specificity has been achievedby conjugating the hematoporphyrin to tumor-specific antibodies. Forexample, when hematoporphyrin was coupled to monoclonal antibodiesdirected to a murine myosarcoma cell line Ml, administration of anti-Mlhematoporphyrin-conjugates to tumor-bearing animals followed by exposureto incandescent light resulted in the suppression of Ml growth (Mew, D.,et al, J Immunol(1983) 130:1473-1477). In additional work,hematoporphyrin was conjugated to a monoclonal antibody specific to anantigen associated with a human leukemia (CAMAL) and the conjugates wereshown to mediate the irradiation-induced killing of leukemic cellsspecifically, in vitro (Mew, D., et al, Cancer Research (1985)45:4380-4386). Conjugation of the related compound chlorine 6 to anti-Tcell Mab has also been reported (Oseroff, A.R., et al.,Proc Natl AcadSci USA (I986) 8]:8744-8748).

While the conjugation of hematoporphyrin to immunoglobulins specific fortargeted cells refines the ability of the hematoporphyrin to home to thedesired cells or tissue, this still does not solve another problemancillary to this general therapeutic approach, namely that thewavelength for irradiation required to activate the hematoporphyrin orHPD, which is in the range of 630 nanometers, is also an energy which isreadily absorbed by the porphyrins and other natural chromophores in theblood and other tissues. Therefore, relatively large amounts of thehematoporphyrin or HPD must be administered, often resulting inoversensitization of the patient to light in general. It would bedesirable to administer compounds to mediate the effects of irradiationin a lower amount, thus avoiding the problems of hypersensitivityexhibited nonspecifically throughout the subject organism. The activityof certain of these compounds was described in a paper by Richter, A.M.,et al, in J Natl Cancer Inst (1987) 79:1327-1332, mailed to subscriberson Jan. 19, 1988. The invention is directed to the use of suchcompounds.

DISCLOSURE OF THE INVENTION

The invention provides light absorbing compounds capable of exhibitinglight-mediated cytotoxic and diagnostic effects. In addition to their invitro use, these compounds may be administered in in vivo relatively lowdosage due to their capability to absorb radiation whose energy range isoutside of that normally absorbed by the components present in highconcentration in the blood or other tissues, in particular, theporphyrin residues normally associated with hemoglobin and myoglobin.Therefore, by providing these modified porphyrins for in vivo treatmentat lower concentration, hypersensitivity of nontarget tissues isreduced, and the irradiation treatment can be conducted at a wavelengthat which the native chromophores do not compete for photons with theactive compounds, resulting in greater depth of penetration of thelight. Similar advantages accrue in in vitro treatment of coloredmaterials, such as blood samples.

These photoactive compounds are modified porphyrins which, by virtue oftheir derivatization, undergo a shift in absorption maxima so that theyappear green rather than red, indicating their absorption of wavelengthsin the red-orange range. This collection of derivatives has thereforebeen nicknamed "green porphyrin" (Gp) and has been shown to confersensitivity on target cells at concentrations greater than 10-fold lowerthan those required for hematoporphyrin (Hp) or HPD.

The Gp is selected from a group of porphyrin derivatives obtained usingDiels-Alder reactions of acetylene derivatives with protoporphyrin underconditions which effect a reaction at only one of the two availableconjugated, nonaromatic diene structures present in theprotoporphyrin-IX ring system (rings A and B). The formulas shown inFIG. 1 represent the green porphyrins of the invention. Also, forconvenience, an abbreviation of the term hydro-monobenzoporphyrinderivative--"BPD"--is generally used to refer to compounds of formulas 3and 4 of FIG. 1, as these are the preferred forms of Gp.

Furthermore, dimeric forms of the Gp can be provided, thus amplifyingthe ability of the Gp compound to absorb light on a per mole basis.Dimeric and multimeric forms of Gp/porphyrin combinations can also beemployed, providing additional absorption wavelengths.

In addition, the modified porphryins (referred to as "green porphyrin"or "Gp" herein) of the invention can be conjugated to specific ligandsreactive with a target, such as receptor-specific ligands orimmunoglobulins or immunospecific portions of immunoglobulins,permitting them to be more concentrated in a desired target tissue orsubstances. This conjugation permits further lowering of the requireddose levels since the material is not wasted in distribution into othertissues whose destruction, far from being desired, must be avoided.

Thus, in one aspect, the invention relates to methods of locating oreffecting cytotoxicity, i.e. photosensitizing, with respect to targetmaterials using the hydro-monobenzoporphyrins of the invention eitheralone or as conjugates. The hydro-monobenzoporphyrins are greenporphyrins (Gp) as shown in FIG. 1, and are localized specifically invivo to certain target tissues, where their presence can be detected byfluorescence, or by other means when the Gp is provided with additionalor alternate labeling. As indicated above, the specificity of the Gp canbe further enhanced by conjugation to ligands specific for the target.In addition, when the Gp is irradiated in situ using light in the rangeof 670-780 nm, photoactivation results in cytotoxicity to thesurrounding tissue. Cells to which the Gp is normally attracted includetumor cells, and neoplastic cells in general, as well as bacteria andother diseased tissues. The method can be applied either in vivo or invivo, and, when applied in vivo, can be topical or systemic.

In another aspect, the invention relates to certain specific Gpcompounds including those of formulas 3 and 4 designated herein "BPD",that are partially hydrolyzed forms containing free (non-esterified)carboxylic acid moieties or their salts in the R³ substituents. Theinvention also relates to labeled forms of these compounds.

In other aspects, the invention relates to conjugates of the formulasRe*-L-Gp and Ig-L-Gp wherein Re* represents a ligand which is specificto, and capable of, binding a receptor at a cell surface, Ig representsan immunoglobulin or an immunologically reactive portion thereof, Gprepresents a hydro-monobenzoporphyrin having an absorption maximum inthe range of 670-780 nanometers, and L represents either a covalent bondlinking these components or a linking moiety covalently linked to eachof the Re* or Ig and Gp.

The invention is also directed to tripartite complexes which includeRe*-L-Gp or Ig-L-Gp further conjugated to or associated with a label.The label may be bound either to the targeting component or to the Gp orboth.

In another aspect, the invention relates to pharmaceutical compositionscontaining these active ingredients.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-2 to 1-6 show the structure of green porphyrin (Gp) compoundsused in the methods and conjugates of the invention.

FIGS. 2-2 to 2-4 show the structure of four preferred forms of thehydro-monobenzoporphyrin derivative of formulas 3 and 4 (BPD).

FIG. 3 shows a comparative absorption spectrum of a BPD compound andprior art compositions.

FIG. 4 shows the results of skin sensitivity assay using a BPD compound.

MODES OF CARRYING OUT THE INVENTION The Hydro-monobenzoporphvrins (Gp)

All of the compositions of the invention employ as the light absorbingcompound, a derivative of the protoporphyrin ring system which has alight absorption maximum in the range of 670-780 nanometers. FIG. 3shows the absorption spectrum of one of the compounds of the inventionshown in FIG. 2, BPD-DA, wherein R¹ and R² are carbomethoxy, incomparison to HPD and Photofrin® II compositions. Only BPD-DA has amajor absorption peak at about 685 nm.

In general, this shift is achieved by effectively saturating one of thetwo π-bonds in one, but not two, of the four pyrrole rings whichconstitute the typical porphyrin system. In protoporphyrin-IX two of thepyrroles contain vinyl substitutions such that the exocyclic π-bond isconjugated to one of the two π-bonds in the ring. A Diels-Alder reactioninvolving one of these conjugated systems with an acetylene derivativedienophile results in a fused cyclohexadiene--referred to herein as"hydrobenzo"--fused to the A or B ring, as shown in formulas 1 and 2.Rearrangement of the π system in the hexadiene ring results in thecompounds of FIGS. 3 and 4; reduction provides the compounds of formulas5 and 6. All of these compounds provide the desired shift in absorptionmaximum.

Specific preparation of some compounds useful in the invention or theirprecursors is described by Morgan, A.R., et al, J Chem Soc Chem Commun(1984) pp. 1047-1048; and by Pangka, B.S. et al, J Organic Chem (1986)51:1094. As described in these publications, it had earlier beenreported that protoporphyrin-IX dimethyl ester, when reacted with strongDiels-Alder dienophile reagents such as tetracyanoethylene, isderivatized to the hydro-dibenzo derivatives. However, it is clear that,as shown by these references, when acetylene is derivatized with moreweakly electron withdrawing groups and used as a Diels-Alder reagent,hydro-monobenzo derivatives are formed. Thus, there are obtaineddirectly from reaction of protoporphyrin with, for example dimethylacetylene dicarboxylate (DMAD), compounds shown as formulas 1 and 2 ofFIG. 1, wherein R¹ and R² represent the substituents on the originalacetylene-derived Diels-Alder reagent, R¹ C═CR² --in this case,carbomethoxy. R¹ and R² are, generally, specifically carbalkoxy groupssuch as carbomethoxy or carboethoxy. R³ represents substituents presenton the porphyrin used in the reaction or substituents derived therefrom.In the Morgan reference, the reaction substrate was protoporphyrin-IXdimethyl ester; thus the ligand R³ was, in all cases,2-carbomethoxyethyl.

The disclosed substituents in the Morgan and Pangka references for theacetylene-derived dienophile include phenylsulfonyl--i.e., SO₂ Ph,either as a single substituent, as described in the foregoing references(β-phenylsulfonylpropiate) or, putatively, wherein both R¹ and R² aresulfonyl derivatives. In general, R¹ and R² are each, independently,moderate electron-withdrawing substituents, and are, most commonly,carbalkoxy, or alkyl or aryl sulfonyl, or any other activatingsubstituents, which are not sufficiently electron-withdrawing to resultin reaction with both A and B rings rather than reaction with only one,such as cyano or --CONR⁵ CO--wherein R⁵ is aryl or alkyl. One of R¹ andR² may optionally be H while the other is an electron withdrawingsubstituent of sufficient strength to facilitate the Diels-Alderreaction.

As used herein, carboxy is, as conventionally defined, --COOH andcarbalkoxy is --COOR, wherein R is alkyl; carboxyalkyl refers to thesubstituent --R'--COOH wherein R6 40 is alkylene; carbalkoxyalkyl refersto --R'--COOR wherein R' and R are alkylene and alkyl respectively.Alkyl is a saturated straight or branched chain hydrocarbyl of 1-6carbon atoms such as methyl, n-hexyl, 2-methylpentyl, t-butyl, n-propyl,and so forth. Alkylene is as alkyl except that the group is divalent.Aryl or alkyl sulfonyl moieties have the formula SO₂ R wherein R isalkyl as above-defined, or is aryl, wherein aryl is phenyl optionallysubstituted with 1-3 substituents independently selected from halo(fluoro, chloro, bromo or iodo), lower alkyl (1-4C) or lower alkoxy(1-4C). In addition, one or both R¹ of R² can itself be aryl --i.e.,phenyl optionally subsituted as above-defined.

As shown in FIG. 1, the adduct formed by the reaction of R¹ --C.tbd.C--R² with the protoporphyrin-IX ring system (R³ is a protected form of2-carboxyethyl such as 2-carbomethoxyethyl or 2-carboethoxyethyl; R⁴ isCH═CH₂) are compounds of the formulas 1 and 2 wherein the compound informula 1 results from addition to the A ring and formula 2 results fromaddition to the B ring. In these resulting products of formulas 1 and 2,R⁴ remains CH═CH₂, however this vinyl group is readily 7 derivatized toother embodiments of R⁴ by addition to or oxidation of the vinyl ringsubstituent of ring B in formula 1 or ring A in formula 2. The additionor oxidation products can be further substituted if the addedsubstituents are functional leaving groups--for example --Br may besubstituted by --OH, --OR (R is alkyl 1-6C as above), or --NH₂, --NHR,--NR₂, etc. In preferred embodiments, one of the added substituents ishydrogen, and the other is selected from the group consisting of halo(fluoro, chloro, bromo or iodo), hydroxy, lower alkoxy, amino or anamide, sulfhydryl or an organo-sulfide or can be, itself, hydrogen.Addition to the vinyl group does not appreciably change the absorptionspectrum of the resulting compound. The product of the Markovnikovaddition of water provides a substituent structure analogous to thehematoporphyrin ring system at the relevant ring. Thus, the compounds ofthe invention include various groups as R⁴, including substituents whichprovide additional prophyrin or prophyrin-related ring systems, as willbe further described below.

R³ in protoporphyrin-IX is 2-carboxyethyl (--CH₂ CH₂ COOH). However, thenature of R³ (unless it contains a π-bond conjugated to ring π-bond), isordinarily not relevant to the progress of the Diels-Alder reaction orto the effectiveness and absorption spectrum of the resulting product.R³ can thus be, for example, lower alkyl (1-4C), or ω-carboxyalkyl(2-6C) or the esters or amides thereof. The R³ substituent may also besubstituted with halogen as above-defined, or with other nonreactivesubstituents. However, as the convenient starting materials for the Gpcompounds of the invention are the naturally occurring porphyrins, thepreferred substituents for R³ are CH₂ CH₂ COOH or --CH₂ CHR² COOR,whereing R is alkyl (1-6C).

It should be noted that while the nature of the R³ substituent does notordinarily influence the course of the Diels-Alder reaction by alteringthe nature of the diene substrate, derivatization may be necessary topromote the reaction by providing suitable solubility characteristics orto prevent interference with the reaction. Thus, the Diels-Alderreactions described by Morgan et al and by Pangka et al utilized thedimethylester of protoporphyrin-IX as a substrate in order to preventinterference with the reaction by the free carboxyl group and to providesuitable solubility characteristics.

In the BPD compounds of the invention, it has been found advantageous tohydrolyze or partially hydrolyze the esterified carboxy group in --CH₂CH₂ COOR. The hydrolysis occurs at a much faster rate than that of theester groups of R¹, R², and the solubility characteristics of theresulting compounds are more desirable than those of the unhydrolyzedform. Hydrolysis results in the diacid or monoacid products (or theirsalts).

The hydro-monobenzoporphyrins which directly result from the Diels-Alderreaction described in the cited references can also be isomerized astherein described (see Morgan et al and Pangka et al (sucra)) tocompounds of formulas shown as 3 and 4 of FIG. 1 by treatment withsuitable reagents such as triethylamine (TEA) in methylene chloride or1,5-diaza bicyclo [5.4.0]undec-5-ene (DBU). The stereochemistry of theproduct is determined by the choice of reagent.

The depictions of compounds 3 and 4 in FIG. 1 do not show the relativeposition of the exocyclic methyl group (ring A of formula 3 and ring Bof formula 4) with respect to the R² substituent. It has been found bythese authors that rearrangement using TEA gives cis geometry for theangular methyl group and R², while treatment with DBU results in thetrans product. This cis product is evidently kinetically controlledsince treatment of the cis product with DBU results in a furtherrearrangment to trans stereochemistry. Thus, formulas 3 and 4 of FIG. 1show the rearranged products generically, from either TEA or DBUcatalyzed rearrangement in rings A and B respectively.

In addition, the Diels-Alder products can be selectively reduced bytreating with hydrogen in the presence of palladium on charcoal to givethe saturated ring analogs, shown as formulas 5 and 6 in FIG. 1,corresponding to the respective Diels-Alder products of rings A and B.These reduced products are less preferred embodiments, and are lessuseful in the method of the invention than the compounds of formulas1-4.

The description set forth above with respect to the compounds offormulas 1 and 2 concerning derivatization by conversion of theremaining vinyl substituent (R⁴) and with respect to variability of -R³applies as well to the compounds of formulas 3, 4, 5 and 6.

The compounds of formulas 3 and 4 (BPD), and especially those which havehydrolyzed and partially hydrolyzed carbalkoxy groups in R³, are mostpreferred. Compounds of the invention which contain --COOH may beprepared as the free acid or in the form of salts with organic orinorganic bases.

It will be noted that many of the compounds of FIG. 1 contain at leastone chiral center and therefore exist as optical isomers. The conjugatesand methods of the invention include compounds having bothconfigurations of the chiral carbons, whether the compounds are suppliedas isolates of a single stereoisomer or are mixtures of enantiomersand/or diastereomers. Separation of mixtures of diastereomers may beeffected by any conventional means; mixtures of enantiomers may beseparated by usual techniques of reacting them with optically activepreparations and separating the resulting diastereomers.

It should further be noted that the reaction products may be unseparatedmixtures of A and B ring additions, e.g., mixtures of formulas 1 and 2or 3 and 4 or 5 and 6. Either the separated forms--i.e., formula 3 aloneor 4 alone, or mixtures in any ratio may be employed in the methods oftherapy and diagnosis set forth herein.

The name "dihydro"-monobenzoporphyrin describes the direct andrearrangement products of the Diels-Alder reaction of the porphyrin ringsystem with R¹ C═C--R² ; "tetrahydro"-monobenzoporphyrin describes theforegoing reduced products of formulas 5 and 6, and"hexahydro"-monobenzoporphyrin describes the analogs containing theexocyclic "benzo" ring completely reduced. Hydro-monobenzoporphyrin isused generically to include all three classes of oxidation state. Themonobenzoporphyrins per se are outside the scope of the invention astheir absorption maxima do not fall within the required range.

FIG. 2 shows four particularly preferred compounds of the inventionwhich have not been previously described in the art. These compounds arecollectively designated benzoporphyrin derivative (BPD) as they areforms of Gp having the formula 3 or 4. These are hydrolyzed or partiallyhydrolyzed forms of the rearranged products of formula 3 and 4, whereinone or both of the protected carboxyl groups of R³ are hydrolyzed. Theester groups at R¹ and R² hydrolyze relatively so slowly that conversionto the forms shown in FIG. 2 is easily effected.

For purposes of this description, R³ is --CH₂ CH₂ COOR^(3'). As shown inFIG. 2, each R^(3') is H in preferred compound BPD-DA, R¹ and R² arecarbalkoxy, and derivatization is at ring A; BPD-DB is the correspondingcompound wherein derivatization is at ring B. BPD-MA represents thepartially hydrolyzed form of BPD-DA, and BPD-MB, the partiallyhydrolyzed form of BPD-DB. Thus, in these latter compounds, R¹ and R²are carbalkoxy, one R³⁺ is H and the other R^(3') is alkyl (1-6C). Thecompounds of formulas BPD-MA and BPD-MB may be homogeneous wherein onlythe C ring carbalkoxyethyl or only the D ring carbalkoxyethyl ishydrolyzed, or may be mixtures of the C and D ring substituenthydrolyzates. In addition, mixtures of any two or more of BPD-MA, -MB,-DA and -DB may be employed in the method of the invention.

As these hydrolyzed forms of the Diels-Alder product are previouslyundisclosed, the invention is also directed to these compounds. Thus, inanother aspect, the invention is directed to compounds of the formulasshown in FIG. 2 wherein R¹ and R² are as above defined, and R is alkyl(1-6C). Preferred are embodiments wherein R¹ and R² are carbalkoxy,especially carbomethoxy or carboethoxy.

Certain other embodiments wherein R⁴ is other than vinyl or wherein R³is a non-native substituent are also not disclosed in the art and theinvention is directed to them, i.e., the invention is directed to thecompounds shown in FIG. 1 wherein

each R¹ and R² is independently selected from the group consisting ofcarbalkoxy (2-6C), alkyl (1-6C) sulfonyl, aryl (6-10C) sulfonyl, aryl(6-10C); cyano; and --CONR⁵ CO-- wherein R⁵ is aryl (6-10C) or alkyl(1-6C);

each R³ is independently carboxyalkyl (2-6C) or a salt, amide, ester oracylhydrazone thereof, or is alkyl (1-6C); and

R⁴ is CHCH₂, CHOR^(4'), --CHO, --COOR^(4'), CH(OR^(4'))CH₃,CH(OR^(4'))CH₂ OR^(4'), --CH(SR^(4'))CH₃, --CH(NR^(4') ₂)CH₃,--CH(CN)CH₃, --CH(COOR^(4'))CH₃, --CH((OOCR^(4'))CH₃,--CH(halo)CH₃, or--CH(halo)CH₂ (halo),

wherein R^(4') is H, alkyl (1-6C) optionally substituted with ahydrophilic substituent, or

wherein R⁴ is an organic group of <12C resulting from direct or indirectderivatization of vinyl, or

wherein R⁴ is a group containing 1-3 tetrapyrrole-type nuclei of theformula -L-P as herein defined;

wherein when R⁴ is CHCH₂, both R³ cannot be 2-carbalkoxyethyl.

Compounds of the formulas 3 and 4 and mixtures thereof are particularlypreferred. Also preferred are those wherein R¹ and R² are the same andare carbalkoxy, especially carboethoxy; also preferred are those whereinR⁴ is --CHCH₂, CH(OH)CH₃ or --CH(halo) CH₃, or is a group containing 1-3tetrapyrrole-type nuclei of the formula -L-P (defined below).

As used herein, "tetrapyrrole-type nucleus" represents a four-ringsystem of the skeleton: ##STR1## and a salt, ester, amide oracylhydrazone thereof, which is highly conjugated. It includes theporphyrin system, which is, in effect, a completely conjugated system,the chlorin system, which is, in effect, a dihydro form of theporphyrin, and the reduced chlorin system, which is a tetrahydro form ofthe completely conjugated system. When "porphyrin" is specified, thecompletely conjugated system is indicated; Gp is effectively a dihydroform of the porphyrin system.

One group of compounds of the invention is that wherein the substituentR⁴ includes at least one additional tetrapyrrole-type nucleus. Theresulting compounds of the invention are dimers or oligomers in which atleast one of the tetrapyrrole-type ring systems is Gp. Linkage betweenthe Gp moiety through the position of R⁴ to an additionaltetrapyrrole-type ring system may be through an ether, amine or vinyllinkage. Additional derivatization in the case of porphyrin ring systemswhich have two available substituent positions (in both A and B rings)corresponding to R⁴ can also be formed, as further described below.

As stated above, the compounds of formulas shown in FIG. 1 include thosewherein the embodiment of R⁴ is formed by addition to the vinyl groupsof initial Gp products. Thus, R⁴ can be any substitutent consistent withthat formed by a facile addition reaction. Thus, both added substituentscan be, for example, OH or halo, and these substituents can be furthersubstituted, or the addition reagent may be of the form HX wherein H isadded to the ring-adjacent carbon to provide R⁴ of the form ##STR2##

The vinyl group can also be oxidized to obtain R⁴ as CH₂ OH, --CHO, orCOOH and its salts and esters.

Thus, in general R⁴ represents any substituents to which the vinyl group--CH═CH₂ is readily converted by cleavage or addition, and furtherresultants of reaction of leaving groups with additional moieties.Typical R⁴ substituents include: ##STR3## CH(OH)Me, --CHBrMe,--CH(OMe)Me, --CH(pyridinum bromide)Me, --CH(SH)Me and the disulfidethereof, --CHOHCH₂ OH, --CHO, and --COOH or --COOMe.

When R⁴ is -L-P, the substituent formula "-L-P" represents a substituentwherein -L- is selected the group consisting of ##STR4## and P isselected from the group consisting of Gp wherein Gp is of the formula1-6 shown in FIG. 1, but lacking R⁴ and conjugated through the positionshown in FIG. 1 as occupied by R⁴ to L, and a porphyrin of the formula##STR5## wherein R³ and R⁴ are as above-defined, and the unoccupied bondis then conjugated to L. It is understood that the abbreviation ##STR6##represents a porphyrin of the formula: ##STR7##

(It is also understood that when -L- is of the formula (e) or (f), thering system to which the double bond is attached will have a resonancesystem corresponding to ##STR8## in the ring to which the double bond isattached, as shown.) ##STR9## wherein R⁴ is as above defined. Thus,compounds of the invention include: ##STR10## and the like.

Preparation of the Dimers and Oligomers

The dimers and oligomeric compounds of the invention can be preparedusing reactions analogous to those for dimerization and oligomerizationof porphyrins per se.

For formation of compounds of the invention where -L- is of the formula##STR11## i.e., an ether linkage, the Gp vinyl group is converted to thehalide, preferably the chloride, by treating the Gp in a solution of,for example, methylene chloride with HBr to recover the additionproduct. The resulting product is harvested by evaporation in vacuo.redissolved in methylene chloride and added to an insoluble base such assolid potassium carbonate. To this is added an equivalent of thetetrapyrrole-type nucleus "P" to be linked wherein the reactive R⁴moiety of "P" is 1-hydroxyethyl. The mixture is stirred for theappropriate amount of time, around 12 hours, generally, and theresulting diastereomeric pair of dimers (the enantiomeric paired formand a meso form) can be separated from the mixture chromatographically.The tetrapyrrole-type nucleus represented by "P" in this procedure canbe either another Gp or a porphyrin.

If the "P" substitutent is a porphyrin, an additional vinyl group may bemade available for further halogenation and further reaction to formhigher order oligomers.

For embodiments wherein -L- contains a vinyl 15 group, the dimers areobtained by treating Gp wherein R⁴ is 1-hydroxyethyl with an equivalentamount of the linking tetrapyrrole-type nucleus also having the linkingR⁴ as 1-hydroxyethyl with a strong, nonnucleophilic acid, such astrifluoromethyl sulfonic acid. This treatment results in precipitationof the resulting methylpropenyl linked dimer. (The ether-linked dimercan be formed as a side product in this reaction by substitutingalternative acids such as sulfuric acid.)

The amino-linked compounds can be formed by treatment of the vinyl groupwith HBr followed by treatment with the appropriate amine to obtain thedesired linkage.

The Tarcet-Specific Component

The target-specific component can be, for example, an immunoglobulin orportion thereof or a ligand specific for receptor.

The immunoglobulin component can be any of a variety of materials. Itmay be derived from polyclonal or monoclonal antibody preparations andmay contain whole antibodies or immunologically reactive fragments ofthese antibodies such as F(ab')2, Fab, or Fab' fragments. Use of suchimmunologically reactive fragments as substitutes for whole antibodiesis well known in the art. See, for example Spiegelberg, H.L., in"Immunoassays in the Clinical Laboratory" (1978) 3:1-23.

Polyclonal anti-sera are prepared in conventional ways by injecting asuitable mammal with antigen to which antibody is desired, assaying theantibody level in serum against the antigen, and preparing anti-serawhen the titers are high. Monoclonal antibody preparations may also beprepared conventionally such as by the method of Koehler and Milsteinusing peripheral blood lymphocytes or spleen cells from immunizedanimals and immortalizing these cells either by viral infection, byfusion with myelomas, or by other conventional procedures, and screeningfor production of the desired antibodies by isolated colonies. Formationof the fragments from either monoclonal or polyclonal preparations iseffected by conventional means as described by Spiegelberg, H.L., supra.

Particularly useful antibodies exemplified herein include the monoclonalantibody preparation CAMAL-1 which can be prepared as described byMalcolm, A., et al, Ex Hematol (1984) 12:539-547; polyclonal ormonoclonal preparations of anti-Ml antibody as described by Mew, D., etal, J Immunol (1983) 130:1473-1477 (supra) and B16G antibody which isprepared as described by Maier, T., et al, J Immunol (1983) 131:1843;Steele, J.K., et al, Cell Immunol (1984) 90:303.

The foregoing list is exemplary and certainly not limiting; once thetarget tissue is known, antibody specific for this tissue may beprepared by conventional means. Therefore the invention is applicable toeffecting toxicity against any desired target.

The ligand specific for receptor, Re*, refers to a moiety which binds areceptor at cell surfaces, and thus contains contours and chargepatterns which are complementary to those of the receptor. The ligandspecific for receptor is symbolized in the formulas of the compounds ofthe invention as Re*, wherein the asterisk indicates that the moietybound in the compound of the invention is not the receptor itself, but asubstance complementary to it. It is well understood that a wide varietyof cell types have specific receptors designed to bind hormones, growthfactors, or neurotransmitters. However, while these embodiments ofligands specific for receptor are know and understood, the phrase"ligand specific for receptor" , as used herein, refers to anysubstance, natural or synthetic, which binds specifically to a receptor.

Examples of such ligands include the steroid hormones, such asprogesterone, estrogens, androgens, and the adrenal cortical hormones;growth factors, such as epidermal growth factor, nerve growth factor,fibroblast growth factor, and so forth; other protein hormones, such ashuman growth hormone, parathyroid hormone, and so forth; andneurotransmitters, such as acetylcholine, serotonin, and dopamine. Anyanalog of these substance which succeeds in binding to the receptor isalso included.

Linkage

The conjugation of the target-cell-specific component to thehydro-monobenzoporphyrin can be effected by any convenient means. Forproteins, such as Ig and certain Re*, a direct covalent bond betweenthese moieties may be effected, for example, using a dehydrating agentsuch as a carbodiimide, in which case L represents a covalent bond. Aparticularly preferred method of covalently bindinghydro-monobenzoporphyrins ,) to the immunoglobulin moiety is treatmentwith 1-ethyl-3-(3-dimethylamino propyl) carbodiimide (EDCI) in thepresence of a reaction medium consisting essentially of dimethylsulfoxide (DMSO). A preparation using this preferred procedure isillustrated in Example 3 below.

Of course, other dehydrating agents such as dicyclohexylcarbodiimide ordiethylcarbodiimide could also be used as well as conventional aqueousand partially aqueous media.

Nonprotein receptor ligands can be conjugated to the Gp according totheir relevant functional groups by means known in the art.

The active moieties of the conjugate may also be conjugated throughlinker compounds which are bifunctional, and are capable of covalentlybinding each of the two active components. A large variety of theselinkers is commercially available, and a typical list would includethose found, for example, in the catalog of the Pierce Chemical Co.These linkers are either homo or heterbifunctional moieties and includefunctionalities capable of forming disulfides, amides, hydrazones, and awide variety of other linkages.

Other linkers include polymers such as polyamines, polyethers, polyaminealcohols, derivatized to the components by means of ketones, acids,aldehydes, isocyanates, or a variety of other groups.

The techniques employed in conjugating the active moieties of theconjugate include any standard means and the method for conjugation doesnot form part of the invention. Therefore, any effective technique knownin the art to produce such conjugates falls within the scope of theinvention, and the linker moiety is accordingly broadly defined only asbeing either a covalent bond or any linker moiety available in the artor derivable therefrom using standard techniques.

Label

For use in the method of the invention either the green porphyrincompounds per se or the conjugates may be further derivatized to acompound or ion which labels the drug. A wide variety of labelingmoieties can be used, including radiosotopes, chromophores, andfluorescent labels. Radioisotope labeling is preferred, as it can bereadily detected in vivo.

The compounds which are Gp alone or are conjugates of Gp with a specificbinding substance can be labeled with radioiostopes by coordination of asuitable radioactive cation in the porphyrin system. Useful cationsinclude technetium, gallium, and indium. In the conjugates, either orboth the specific binding substances can be linked to or associated withlabel, or the label can be conjugated or coordinated with the Gp moietyitself.

Metal Ions

The compounds of the invention can be administered or used in in vitromethods as shown above or when complexed to appropriate metal ions. Asis generally understood in the art, the tetrapyrrole-type nucleus can betreated with an appropriate ion such as magnesium ion, zinc ion,stannous ion, and the like to obtain the metal complex. As stated above,the metal ion may also be a radiolabel. The nature and desirability ofthe inclusion of a metal ion in the tetrapyrrole-type nucleus depends onthe specific application for which the compound is intended. When theinclusion of a metal ion is desired, the desired metal ion can beinserted using the appropriate metal salts under known conditions. Forexample, zinc ion can be introduced by treating the compound with zincacetate in 1:1 methylene chloride:methanol.

Administration and Use

The improved photosensitizing compounds of the invention are thus usefulin general, in the manner known in the art for hematoporphyrinderivative and for DHE. These materials are useful in sensitizingneoplastic cells or other abnormal tissue to destruction by irradiationusing visible light--upon photoactivation, the compounds have no directeffect, nor are they entered into any biological event; however theenergy of photoactivation is believed to be transferred to endogenousoxygen to convert it to singlet oxygen. This singlet oxygen is thoughtto be responsible for the cytotoxic effect. In addition, thephotoactivated forms of porphyrin fluorescence which fluoresce can aidin localizing the tumor.

Typical indications, known in the art, include destruction of tumortissue in solid tumors, dissolution of plaques in blood vessels (see,e.g., U.S. Pat. No. 4,512,762); treatment of topical conditions such asacne, atheletes foot, warts, papilloma, and psoriasis and treatment ofbiological products (such as blood for transfusion) for infectiousagents, since the presence of a membrane in such agents promotes theaccumulation of the drug.

The conjugate of the invention, of the hydro-monobenzoporphyrins whenemployed alone are formulated into pharmaceutical compositions foradministration to the subject or applied to an in vitro target usingtechniques known in the art generally. A summary of such pharmaceuticalcompositions may be found, for example, in Remington's PharmaceuticalSciences. Mack Publishing Co., Easton, PA, latest edition.

The conjugates or compounds of the invention taken alone can be used inthe systemic treatment of tumors and neoplastics made as bronchial,cervical, esophageal or colon cancer and for the diagnosis of same.

The conjugates and hydro-monobenzoporphyrins of the present invention,labeled or unlabeled, can be administered systemically, in particular byinjection, or can be used topically. The Gp or conjugates can be usedsingly or as components of mixtures.

Injection may be intravenous, subcutaneous, intramuscular, or evenintraperitoneal. Injectables can be prepared in conventional forms,either as liquid solutions or suspensions, solid form suitable forsolution or suspension in liquid prior to injection, or as emulsions.Suitable excipients are, for example, water, saline, dextrose, glyceroland the like. 0f course, these compositions may also contain minoramounts of nontoxic, auxiliary substances such as wetting or emulsifyingagents, pH buffering agents and so forth.

Systemic administration can also be implemented through implantation ofa slow release or sustained release system, by suppository, or, ifproperly formulated, orally. Formulations for these modes ofadministration are well known in the art, and a summary of such methodsmay be found, for example, in Remington's Pharmaceutical Sciences(supra).

For diagnosis, the compounds may be used along or may be labeled with aradiosotope or other detecting means.

If treatment is to be localized, such as for the treatment ofsuperficial tumors or skin disorders, the active conjugates orhydro-monobenzoporphyrins may be topically administered using standardtopical compositions involving lotions, suspension, or pastes.

The quantity of conjugates or green porphyrin derivative to beadministered depends on the choice of active ingredient, the conditionto be treated, the mode of administration, the individual subject, andthe judgment of the practitioner. Depending on the specificity of thepreparation, smaller or larger doses may be needed. For compositionswhich are highly specific to target tissues, such as those whichcomprise conjugates of the green porphyrin with a highly specificmonoclonal immunoglobulin preparation or specific receptor ligand,dosages in the range of 0.05-1 mg/kg are suggested. For compositionswhich are less specific to the target tissue, larger doses, up to 1-10mg/kg may be needed. The foregoing ranges are merely suggestive, as thenumber of variables in regard to an individual treatment regime is largeand considerable excursions from these recommended values are expected.

In addition to in vivo use, the compounds of the invention can be usedin the treatment of materials vitro to destroy harmful viruses orinfectious agents. For example, blood plasma or blood which is to beused for transfusion or banked for future transfusion can be treatedwith the compounds of the invention and irradiated to effectsterilization. In addition, biological products such as Factor VIIIwhich are prepared from biological fluids can be irradiated in thepresence of the compounds of the invention to destroy contaminants.

EXAMPLES

The following example are intended to illustrate the invention but notto limit its scope.

EXAMPLE 1 In Vitro Photosensitization by Green Porphyrins

Target cells were washed three times in serum-free medium (DME), countedand made up to a concentration of 10⁷ cells per ml.

For the "affinity" assay, in the dark, 100 μl of the target cellsuspension and 100 μl of the test or control compound were mixed."Labeling" was allowed to continue for one hour at 4° C, and labeledcells were washed in the dark three times with 3 ml medium each time andresuspended in fresh medium. The resuspended cells were then subjectedto light exposure at 300-750 nanometers for 30 minutes.

In a "direct" assay the target cells were irradiated immediately uponaddition of the test or control compound.

The effect of irradiation was estimated using methods appropriate to thetarget cells.

When human erythrocytes (RBCs) were used as target cells, the hemolysiscaused by irradiation of control (hematoporphyrin, Hp) labeled and greenporphyrin (Gp) labeled cells were estimated visually. The Gp used inthis Example was the BPD-DB of FIG. 2 wherein R¹ and R² are carboethoxy.Repeated tests showed this green porphyrin to be 20-30 times more activethan Hp in this assay. Thus, a concentration of 250 ng/ml Hp wasrequired under the above conditions to obtain 50% hemolysis while only10 ng/ml of green porphyrin was required to hemolyze 50% of the RBCs.

When the murine mastocytoma cell line P815 was used, the results weredetermined as follows:

The cells were labeled as above using concentration of 10-50 ng/ml of Hpas control and the BPD-DB as the test substance. The resuspended cellswere treated with 300-750 nm light for 30 minutes and the viabilityresulting was estimated by direct counting using eosin-Y exclusion, astandard procedure for differentiating living from dead cells.

In other determinations conducted as above, the cells recovered fromlight exposure were assayed for L viability by incubating them for 18hours in 10 μCi/ml tritium-labeled thymidine according to the standardprocedure whereby thymidine incorporation is equated with viability. Thecells were harvested and radioactivity uptake was measured by ascintillation counter.

Fifty percent of the P815 cells were killed at 580 ng/ml Hp, but at only32 ng/ml green porphyrin (BPD-DB).

The results of each determination on a variety of cells is shown inTable 1 (LD⁵⁰ in the concentration of compound required to kill 50% ofthe cell population.)

                  TABLE 1                                                         ______________________________________                                                     LD.sub.50 (ng/ml)                                                             Direct test    Affinity test                                     Cell line      Gp      Hp       Gp    Hp                                      ______________________________________                                        Normal lymphocytes                                                                             4.2    31      11      100                                   HL-60            3.5    64        7.2   145                                   K562           70      770      33    2,500                                   KG-1           163     960      80    2,350                                   P815           32      580      26    1,300                                   ______________________________________                                    

EXAMPLE 2 Selective Binding of Green Porphyrin

P815 cells were incubated as described in Example 1 using 1-200 ng/ml Hpor Gp. The Gp was BPD-DB of FIG. 2 wherein R¹ and R² are carboethoxy.The cells were labeled in the dark for 30 minutes, washed free ofunabsorbed porphyrins, resuspended, and then exposed to 300-750 nm lightfor another 30 minutes. Viability of the cells was established bytritiated thymidine incorporation after labeling with 20 μCi/mltritiated thymidine and incubating at 37° C. for 18 hours.

The results showed that 50% of the P815 cells were destroyed at 6-20ng/ml BPD-DB or at 200 ng/ml hematoporphyrin.

EXAMPLE 3 Preparation of Immunoconjugates

This example describes methods of preparation for immunoconjugates offour different anitibody preparations with either hematoporphyrin (Hp)or green prophyrin (Gp); in this example, Gp is BPD-DB of FIG. whereinR¹ and R² are carboethoxy. The antibodies employed were CAMAL-1, anti-Mlantibody, and B16G antibody, all prepared as described hereinabove, andaffinity purified rabbit/anti-mouse Ig (RαMIg). In addition, a purifiedirrelevant monclonal preparation (C-MAb) was used where a control wasdesired.

One preparation of the conjugates is basically as described in Mew, D.,et al, J Immunol (1983) 130:1473 (supra). Briefly, to 220 mg pH 0.2 HCl(Sigma Chemical Co., St. Louis, MO) in 25 ml water and 0.8 mlN,N-dimethylformamide was added 20 mg1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide HCl (EDCI) in 0.6 mlwater. After 30 minutes, this solution was mixed with 15 mg of theantibody protein dissolved in 5 ml distilled water and incubated for 5hours. During this period, the pH of the solution was monitored andadjusted to between 6 and 7. Then 50 μl of monoethanolamine were added,and the solution was allowed to stand overnight at room temperature. Thesolution was dialyzed against 0.001 M phosphate buffer pH 7.4 for fourdays with three changes per day and overnight against PBS. The conjugateof green porphyrin is analogously prepared.

In a preferred method, the conjugation is conducted in an entirelynonaqueous solvent.

In a typical protocol, 2 ml of a dispersion in DMSO containing 5 mg eachof the Hp or Gp and the dehydrating agent is prepared and stirred for 30minutes at room temperature under nitrogen. To this is added adispersion containing 2 mg of the appropriate immunoglobulin in 2 ml ofDMSO, and the resulting mixture stirred for another 10 minutes. Thismixture is then worked up by dilution in phosphate-buffered saline, pH7.4 (PBS) by adding 5 times the volume of PBS containing 50 μlmonoethanolamine, and is then dialyzed against PBS using three changesof wash.

Alternatively, 2 ml of a dispersion containing 5 mg each of Hp or Gp, alinking agent, and a dehydrating agent is prepared and stirred forapproximately 15 minutes at room temperature under nitrogen. To this isthen added a dispersion containing about 2 mg of the immunospecificprotein in 2 ml of tetrahydrofuran and the resulting mixture stirred foranother 10 minutes. The mixture is then worked up as described above.

The foregoing procedures are appropriate for CAMAL-1 and for theremaining antibody preparations above listed.

In addition, the following preparations were made specifically with B16Gand RαMIg:

B16G

11 mg of hematoporphyrin plus 11 mg EDCI in 4 ml spectral grade DMSO wasstirred for 30 minutes under nitrogen at room temperature before theaddition of 20 mg lyophilized B16G antibodies, prepared as described byMaier, T., et al, J Immunol (1983) 131:1843, in 2 ml DMSO. The resultingmixture was stirred for 40 seconds at room temperature and worked up asdescribed above. The resulting product contained 375 μg Hp/mg B16G. Asimilar procedure is used substituting Gp for Hp.

RαMIg

400 μg of EDCI and 400 μg hematoporphyrin in 1 ml DMSO were stirred for30 minutes under nitrogen at room temperature as above before theaddition of 800 μg lyophilized RαMIg antibodies, prepared as describedby Mew, D., et al, J Immunol (1983) 1473-1477, in 1 ml DMSO. Theresulting mixture was stirred for 30 seconds and worked up as describedabove to obtain a product containing 200 μg Hp/mg RαMIg. A similarprocedure is used substituting Gp for Hp.

EXAMPLE 4 Specificity of Immunoconjugates in Vitro

In the following determinations, the levels of antibody conjugation wereas follows, expressed as μg Hp or green porphyrin (Gp) per mgimmunoglobulin:

RαMIg-Hp: 110 μg/mg;

B16G-p, 156 μg/mg;

CAMAL-1-Hp, 260 μg/mg;

Anti-Ml-Hp, 170 μg/mg;

C-MAb-Hp, 95 μg/mg;

RαMIg-Gp, 120 μg/mg;

B16G-Gp, 165 μg/mg;

CAMAL-1-Gp, 75 μg/mg;

C-MAb-Gp 90 μg/mg.

The Ig-Hp and Ig-Gp conjugates are tested against cells in vivo bymixing the conjugates with the appropriate cell types, along withsuitable controls, and then exposing the labeled cells to irradiation.Procedures for carrying out this assay were described in detail in Mew,D., et al, Cancer Research (1985) for CAMAL-1, and by Mew, D., et al, JImmunol (1983) for Anti-Ml, both references cited hereinabove andincorporated herein by reference.

Briefly, for CAMAL-1, three cell lines, WC4, WC6 and WC2 (WC4 and WC6produces the CAMAL antigen, but WC2 does not), are labeled with theappropriate Ig-Hp or Ig-Gp preparation as described above in Example 1.The labeled cell preparations containing 10⁶ cells each are introducedto Rose chambers and exposed to light activation with a laser at 630 nm.The results for various preparations are then compiled.

For the anti-Ml conjugate, Ml tumor cells are used as target cells andtreated with the Ig-Hp, Ig-Gp conjugates or drug or antibody alone orthe combination of antibody and drug, but uncoupled, by incubating themin 6% CO₂ humidified incubator at 37° for two hours. The cells arewashed three times in PBS and then plated and exposed to fluorescentlight overnight. The cells are assessed for viability by tritiatedthymidine uptake as above.

For the B16G conjugates, A10, P815, and L1210 cells are used as targetcells. (A10 cells are a T-cell hybridoma which secretes a B16G-reactiveT suppressor factor; P815 cells are also reactive with B16G.) The invitro study is done using a direct method employing the B16G-Hp orB16G-Gp conjugate or indirectly using unlabeled B16G antibodies andlabeled RαMIg-Hp or RαMIg-Gp.

In a direct method, 5×10⁵ cells are suspended in 1 ml DME/Hepescontaining the appropriate Ig-drug conjugate as test or control at Hp orGp concentrations of 320, 160, 80, 40 and 20 ng drug/ml. The cells areincubated in the dark at 37° for one hour, then washed three times in 5ml DME/Hepes and then resuspended in 1 ml of the same buffer. Three 100μl test portions of the labeled preparations are dispensed into flatbottom microtiter wells and the remainder of the cell suspensions (700μl ) are exposed to incandescent light (22.5 mW/cm²) at a distance of 20cm for one hour. Then three additional 100 μl aliquots are removed tomicrotiter wells. Tritium-labeled thymidine diluted in DME/Hepescontaining 20% FCS is then added to all microtiter wells in 100 μlaliquots so that 2 μCi of labeled thymidine is added to each well.Cultures are incubated for 18 hours at 37° C. and humidified 10% CO₂ andthen harvested on a MASH harvester. Thymidine incorporation was measuredwith an Hp scintillation counter (Tri-Carb Model 4550). The results ofthis study for Ig-Hp are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                                 % killing of cell lines                                              (ng Hp/ml) A10          P815   L1210                                          ______________________________________                                        B16G Hp                                                                       320        100          70     55                                             160        100          50     10                                             80         100          20     0                                              40         65           10     0                                              20         20            0     0                                              C-Mab-Hp                                                                      320        63           75     50                                             160        35           48     15                                             80          0           25     0                                              40          0           12     0                                              20          0            0     0                                              ______________________________________                                    

In an indirect assay, the A10 suspended cells, prepared as describedabove, are exposed to 50 μg/ml of either B16G or a control antibodyC-Mab at 4° C. for 30 minutes, washed in DME/Hepes, and then exposed foran additional 30 minutes at 4° C. in the dark to varying concentrationsof RαMIg-Hp or RαMIg-Gp between 2 μg/ml and 15 ng/ml of Hp or Gp. Thecells are assessed for viability using labeled thymidine uptake asdescribed above. These results for Ig-Hp are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        RαMIg-Hp  Primary antibody                                              (ng/ml)         B16G    C-MAb                                                 ______________________________________                                        500             100     30                                                    250             85      22                                                    125             75      5                                                     52.5            60      2                                                     31.2            47      3                                                     15.6            18        1.5                                                 ______________________________________                                    

Similar results are obtained using corresponding conjugates with Gp.

EXAMPLE 5 In Vivo Cytotoxicity of the Immunoconjugates

The efficacy of the conjugates and of the Gp compounds of the inventionin vivo is also assessed. For the CAMAL-1 and anti-Ml conjugates, theprocedures are as described in the two Mew, et al, papers referencedabove in Example 4. The Gp compound alone shows superior results atappropriate wavelengths as compared to the Hp labeled conjugates.

For the B16G-Hp or B16G-Gp conjugates and for the Gp (BPD-DB) alone, thein vivo studies are conducted as follows:

The in vivo test relies on the indirect effect of a population ofT-suppressor cells on tumors, which then serve as means to assess theeffectiveness of the irradiation treatment. P815 mastocytoma cells grownin syngeneic DBA/2 mice stimulate T-suppressor cells specific for thetumor. These T-suppressor cells impede the development of specificT-killer cells which would otherwise aid in the regression of the tumor.The T-cell hybridoma designated A10 above secretes a T-suppressor factorwhich is associated with these T-suppressor cells. Thus, selectivekilling of these T-suppressor cell populations through reaction withconjugates in which the Ig is an antibody specific for the T-suppressorfactor on the surface of the cells (namely B16G) should result in tumorregression in mice bearing the P815 tumors.

Therefore, in this assay, DBA/2 mice are injected in the right flanksubcutaneously with 10⁴ P815 cells to incorporate the tumor. On dayeight, when the tumors are palpable approx. 25-42 sq mm) the mice arerandomly sorted into groups of eight and injected IV with 150 μl PBScontaining nothing, Hp or Gp, B16G-Hp or B16G-Gp, B16G plus either drug,B16G alone or C-MAbHp or C-MAb-Gp. The levels of Hp are 50 μg per animalin all cases and B16G 310 μg in all cases (where appropriate).

The animals are maintained in the dark for two hours and then exposed tostrong light at 300-750 nm and 2.5 mW/cm² The animals were then treatednormally and monitored on a daily basis.

Animals treated with B16G Hp survived and were tumor free after 100days. Results obtained are shown in Table 4.

                  TABLE 4                                                         ______________________________________                                                    Mean sur-           % tumor-free                                  Experiment  vival time  No. of  after 100                                     Treatment   (days)      cures   days                                          ______________________________________                                        1    PBS        25.0        0/7   0                                                B16G-Hp    41.3        3/9   33                                          2    PBS        23.5        0/6   0                                                Hp         21.0        0/8   0                                                B16G-Hp    24.2        3/8    37.5                                       3    PBS        24.1        0/7   0                                                Hp         23.4        0/7   0                                                B16G + Hp  23.5        0/6   0                                                B16G-Hp    29.2        2/7   29                                          4    PBS        25.2        0/8   0                                                B16G       28.3        0/8   0                                                Hp         24.2        0/8   0                                                B16G + Hp  24.6        0/7   0                                                B16G-Hp    36.7        3/7   43                                          5    PBS        23.8        0/8   0                                                Hp         27.0        0/8   0                                                C-MAb-Hp   20.3        0/8   0                                                B16G-Hp    34.0        1/8    12.5                                       ______________________________________                                    

Similar results are obtained for Gp alone or Gp conjugates.

EXAMPLE 6 In Vitro Evaluation of BPD-DA, -MA, -DB and -MB

The four compounds shown in FIG. 2, wherein R¹ and R² are carbomethoxy,were tested in vitro as described in Example 1. All four compounds werephotosensitive; the monoacid forms BPD-MA and BPD-MB were somewhat moreactive.

EXAMPLE 7

Biodistribution and Decradation

Biodistribution studies have been conducted using tritiated BPD-MA andBPD-MB. Table 5 shows the ratios between ³ H-BPD-MA concentration in thetumor and in normal tissues determined at various times post-injectionin mice bearing P815 tumor as the average for 3 mice.

                  TABLE 5                                                         ______________________________________                                               Time Post Injection                                                    Tissue   3 h    24 h     48 h 72 h   96 h 168 h                               ______________________________________                                        Blood    0.52   1.45     1.37 1.66   2.77 3.65                                Brain    3.76   3.06     2.92 2.69   4.18 6.91                                Heart    1.09   1.71     1.63 1.46   2.24 2.51                                Intestine                                                                              2.42   1.85     1.88 1.48   3.29 2.23                                Lung     0.79   1.55     1.47 1.16   1.63 1.79                                Muscle   2.68   2.98     2.77 2.16   3.45 4.23                                Skin     2.57   1.64     1.95 1.57   2.03 3.51                                Stomach  1.57   1.89     2.08 2.04   2.23 2.98                                ______________________________________                                    

Tumor skin ratios are most favorable 3 hours after IV administration ofthe drug.

To determine biodegradability, tritiated BPD-MA was injected IV intoP815 tumor-bearing mice. The mice were sacrificed at either 3 or 24hours following injection and tumors, livers and kidneys were removed.The BPD-MA in these tissues was extracted and photoactivity was assessedin P815 target cells as described above in Example 1 under standard invitro conditions. While 100% of BPD-MA in tumor was active at hours,only 39% was active at 24 hours; both the liver and kidney degraded BPDmore rapidly than did tumor tissue. Administration of tritiated BPD-MBin the same system gave similar results.

Similar studies using BPD-MA conjugated to an anti-keratin Mab in amodel murine system carrying the KLN squamous tumor cell line showedimproved concentration of the drug in the target tissue.

EXAMPLE 8 In Vivo Photosensitization by BPD

Studies of potential photosensitizers were performed using the M-1rhabdomycoscercoma system in DBA/J2 mice. The compositions to be testedwere diluted to a concentration of 800 μg/ml in PBS from a stock 25solution in DMSO at 8 mg/ml (except Photofrin® II, which was diluteddirectly from the clinical vial). Animals (8 per group) received 0.1 ml(80 μg) of material IV 24 h prior to exposure to light, provided by a150W tungsten bulb, red filter (transmits light >600 nm), hot mirror(reflects light >720 nm) and 2 fiber optics, at 567 Jo/cm².

The results, shown in Table 6, indicate all BPD compounds tested gavepositive results. The superior results shown by Photofrin® IIcompositions are explainable by the observation that initial tumor sizeswere smaller (a result of chance).

                  TABLE 6                                                         ______________________________________                                                                       Tumor Volume at                                          Days Tumor Number of Time of Light                                  Photosensitizer                                                                         Free (PR)  Cures*    Treatment (mm.sup.3)                           ______________________________________                                        None       0.5       2         22.4 ± 7.8                                  Photofrin ® II                                                                      21.3       5         11.9 ± 6.9                                  composition                                                                   BPD-MA     9.2       4         19.0 ± 13.0                                 BPD-MB    10.6       3         18.2 ± 11.0                                 BPD-DA    10.7       4         18.7 ± 9.9                                  BPD-DB    10.6       3         25.4 ± 16.4                                 ______________________________________                                         *Animals whose tumors regressed and who remained tumorfree for 30 days.  

Similar studies, except using a light dose of 378 To/cm³ resulted in theoutcome shown in Table 7.

                  TABLE 7                                                         ______________________________________                                                  Number of               Number of                                   Photosensitizer                                                                         Animals   Days Tumour-free                                                                            Cures                                       ______________________________________                                        None      11        0.1           2                                           Photofrin II                                                                            10        9.5           4                                           BPD-MA    10        13.2          4                                           BPD-MB     9        8.7           6                                           BPD-DA    15        2.5           4                                           BPD-DB    13        13.0          8                                           ______________________________________                                    

The foregoing results are preliminary, and the assay protocols have notyet been optimized.

EXAMPLE 9 Alternate In Vivo Assay

Mice bearing small tumors were injected IV with drug to be tested. Threehours later the animals were sacrificed and their tumors removed. Thetumor cells were teased apart to form a single cell suspension, and thecells were plated at 10⁵ /well and exposed to light at a prescribeddose. The plates were incubated overnight and assayed for viability byMTT assay.

The results of one study are shown in Table 8.

                  TABLE 8                                                         ______________________________________                                                  Dose         Light Dose                                             Photosensitizer                                                                         (μg/mouse)                                                                              (Jo)      % Kill                                       ______________________________________                                        BPD-MA    33           5.7       22.0                                                   40           3.8       32.5                                                   80           3.8       63.5 ± 2.1                                          80           3.8       53.7 ± 6.2                                BPD-MB    33           5.7       25.2                                         BPD-DA    80           3.8       11.0                                                   80           7.6       26.0                                         ______________________________________                                    

Thus, the BPD forms tested were active in this assay; it appears lightintensity and drug levels are subject to optimization and correlation.

EXAMPLE 10 Comparison of BPD to Photofrin® II Compositions

Mice bearing P815 tumors were shaved and injected with equivalentamounts of photosensitzer, and exposed to 72 Jo/cm² (80 mw/cm² -15min-full spectrum) at various time intervals. Skin biopsies were takenat 24 and 48 hours after light irradiation and visual evaluations weremade blind. The results of these evaluations are shown in FIG. 4. BPD-MAand, to a lesser extent, BPD-MB had major photosensitizing activity,under these conditions; this was only present when light treatment wasgiven 3 hours post drug administration, consistent with thebiodegradability of these compounds.

EXAMPLE 11 Preparation of Compounds of the Invention

The following compounds have been prepared using the above-describedDiels-Alder reaction of MeOOC--C═C--COOMe with the dimethyl ester ofprotophorphyrin IX, followed by rearrangement to the forms shown asformulas 3 and 4 of FIG. 1 and by subsequent treatment to hydrolyze ormodify the propionic ester on rings C and D and/or to modify theunreacted vinyl group on the A or B ring remaining after the Diels-Alderreaction with the B or A ring, as the case may be. The products arecompounds of the following formulas, wherein R^(3") is OR* or NR*wherein R* is alkyl, alkylene, or H (or an organic or unorganic cation):##STR12## wherein R¹ and R² are, in all cases, COOMe.

The compounds prepared are as follows:

    __________________________________________________________________________    R.sup.3" (C)                                                                            R.sup.3" (D) R.sup.4                                                __________________________________________________________________________    A-Ring                                                                         1.                                                                             OMe     OMe          CHCH.sub.2                                              2.                                                                             OH      OMe          CHCH.sub.2 (BPD-MA)                                     3.                                                                             OMe     OH           CHCH.sub.2 (BPD-MA)                                     4.                                                                             OH      OH           CHCH.sub.2 (BPD-DA)                                     5.                                                                             OMe     OMe          CH(NH.sub.2)Me                                          6.                                                                             OMe     OMe                                                                                         ##STR13##                                              7.                                                                             OH      OH                                                                                          ##STR14##                                             B-Ring                                                                         1.                                                                             OMe     OMe          CHCH.sub.2                                              2.                                                                             OH      OMe          CHCH.sub.2                                              3.                                                                             OMe     OH           CHCH.sub.2                                              4.                                                                             OH      OH           CHCH.sub.2                                              5.                                                                             OMe     OMe          CH(NH.sub.2)Me                                          6.                                                                             OH      OH           CH(NH.sub.2)Me                                          7.                                                                             OMe     OMe          CH(NH(CH.sub.2).sub.6 NH.sub.2)CH.sub.3                 8.                                                                             OH      OH           CH(NH(CH.sub.2).sub.6 NH.sub.2)CH.sub.3                 9.                                                                             OCD.sub.3                                                                             OCD.sub.3    CH(NH(CH.sub.2).sub.6 NH.sub.2)CH.sub.3                10.                                                                             OMe     OMe          CH(imidazolyl)CH.sub.3                                   OMe     OMe                                                                                         ##STR15##                                               OMe     OMe                                                                                         ##STR16##                                               OMe     OMe          CH(OH)Me                                                 OMe     OMe          CHBrMe                                                   OMe     OMe          CH(OMe)Me                                                OMe     OMe          CH(pyridinium Br)Me                                      NH(CH.sub.2).sub.6 NH.sub.2                                                           OMe          CHCH.sub.2                                               R.sup.3"                                                                              R.sup.3" NH(CH.sub.2).sub.6 NH                                                             CHCH.sub.2                                               OMe     OMe          CH(SH)CH.sub.3                                         20.                                                                             OMe     OMe          disulfide of above                                       OMe     OMe          CHO                                                      OMe     OMe          CHOHCH.sub.2 OH                                        __________________________________________________________________________

EXAMPLE 12 Preparation of BPD dimer-Vinyl Linked

To a stirring solution of BPD-DB (wherein R¹ ═R² ═carbomethoxy and whichis esterified so that both R³ are carbomethoxyethyl) (35 mg, 48 μmol) in5 ml of dichloromethane cooled to dry ice/acetone temperature was addedtrifluoromethanesulfonic acid (34 μl, 380 μmol). An oil separated outupon the addition of the acid. The reaction was brought up to 0° C. Then5 ml of 5% sodium bicarbonate was added to the reaction to neutralizethe acid. The product distributed into the organic layer which waswashed three times with water. The solvent was removed and the productwas dried via azeotrope with acetonitrile.

Preparative thin layer chromatography on silica gel eluting with 10%ethylacetate/dichloromethane gave a single fraction (28 mg, 80% yield).Parent ion in mass spectrum was 1464. The complex proton NMR due to thenumber of isomeric compounds had the characteristic single vinylhydrogen associated with a C-linkage at about 8.1 ppm.

We claim:
 1. A compound of the formula ##STR17## or the metalated and/orlabeled form thereof; wherein each R¹ and R² is independently selectedfrom the group consisting of carbalkoxy (2-6C), aklyl (1-6C) sulfonyl,aryl (6-10C) sulfonyl, aryl (6-10C); cyano; and --CONR⁵ CO--where R⁵ isaryl (6-10C) or alkyl (1-6C);each R³ is independently carboxyalkyl(2-6C) or a salt, amide, ester or acylhydrazone thereof, or is aklyl(1-6C); and R⁴ is CHCH₂, [CHOR^(4'),]--CH₂ OR^(4') --CHO, --COOR^(4'),--CH(OR^(4'))CH₃,CH(OR^(4'))CH₂ OR^(4'),--CH(SR^(4'))CH₃, --CH(NR^(4')₂) CH₃, --CH(CN)CH₃, --CH(COOR^(4'))CH₃ --CH((OOCR^(4'))CH₃,--CH(halo)CH₃, or --CH(halo)CH₂ (halo), wherein R^(4') is H or alkyl(1-6C) optionally substituted with a hydrophilic substituent, or whereinR⁴ consists of 1-3 tetrapyrrole-type nuclei of the formula -L-P wherein-L- is selected from the group consisting of ##STR18## and P is selectedfrom the group consisting of Gp which is of the formula 1-6 but lackingR⁴ and conjugated through the position shown as occupied by R⁴ to L, anda porphyrin of the formula: ##STR19## wherein two of the bonds shown asunoccupied on adjacent rings are joined to R³ and one of the remainingbonds shown as unoccupied is joined to R⁴ and the other to L; with theproviso that if R⁴ is CHCH₂, both R³ cannot be carbalkoxyethyl.
 2. Acompound of the formula ##STR20## or the metalated and/or labeled formthereof; wherein each R¹ and R² is independently selected from the groupconsisting of carbalkoxy (2-6C), alkyl (1-6C) sulfonyl, aryl (6-10C)sulfinyl, aryl (6-10C); cyano; and --CONR⁵ CO--where R⁵ is aryl (6-10C)or alkyl (1-6C);each R³ is independently carboxyalkyl (2-6C) or a salt,amide, ester or acylhydrazone thereof, or is alkyl (1-6C); and whereinR⁴ is a non-interfering organic group of <12C resulting from direct orindirect derivatization of vinyl.
 3. The compound of claim 1 or 2wherein R¹ and R² are carbalkoxy.
 4. The compound of claim 3 wherein R³and R² are carbomethoxy or carboxethoxy.
 5. The compound of claim 1 or 2wherein each R³ is --CH₂ CH₂ COOH or a salt, amide, ester oracylhydrazone thereof.
 6. The compound of claim 3 wherein each R³ is--CH₂ CH₂ COOH or a salt, amide, ester or acylhydrazone thereof.
 7. Thecompound of claim 1 or 2 which is of formulae 3 or
 4. 8. The compound ofclaim 6 which is of formulae 3 or
 4. 9. The compound of claim 1 or 2whereinwherein R⁴ is a group containing 1-3 tetrapyrrole-type nuclei ofthe formula -L-P.
 10. The compound of claim 8 whereinwherein R⁴ is agroup containing 1-3 tetrapyrrole-type nuclei of the formula -L-P. 11.The compound of claim 1 or 2 which is selected from compounds of theformula ##STR21## wherein R is alkyl (1-6C).
 12. A pharmaceuticalcomposition which is useful in targeting specific biological materialwhich composition comprises an effective amount of the compound of claim1 or 2 in admixture with at least one pharmaceutically acceptableexcipient.